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Fibroblasts were passed onto glass, fixed in 2% paraformaldehyde in PBS for 10 minutes, permeabilized in 2% paraformaldehyde containing 0.1% Triton X100 for 10 minutes and washed three times in PBS containing 0.5% BSA and 0.15% glycine, pH 7.4 (Buffer A). Following a 30 min incubation with purified goat IgG (50 (μg/ml) at 25°C and three additional washes with Buffer A, cells were incubated for 60 min with E2-specific antiserum [30] at 1 μg/ml followed by three washes in Buffer A and 60 minute incubation in fluorescently labeled second antibody (1–2 μg/ml). The cells were then washed six times (5 min/wash) in Buffer A and then mounted in gelvatol (Monsanto, St Louis). When livers from newborn pups were examined, fixation was by immersion in 2% paraformaldehyde followed by cryoprotection and shock freezing in liquid nitrogen cooled isopentane and sectioning (5 microns). Otherwise processing was as for the cells above (without the fixation and permabilization steps)."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T7154","span":{"begin":8,"end":13},"obj":"NCBITaxon:10088"},{"id":"T7155","span":{"begin":14,"end":23},"obj":"UBERON:0000922"},{"id":"T7156","span":{"begin":24,"end":35},"obj":"CL:0000057"},{"id":"T7157","span":{"begin":55,"end":64},"obj":"UBERON:0000922"},{"id":"T7158","span":{"begin":107,"end":118},"obj":"CL:0000057"},{"id":"T7159","span":{"begin":155,"end":171},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T7160","span":{"begin":215,"end":231},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T7161","span":{"begin":248,"end":259},"obj":"CHEBI:9750"},{"id":"T7162","span":{"begin":335,"end":342},"obj":"CHEBI_SO_EXT:glycine"},{"id":"T7163","span":{"begin":352,"end":358},"obj":"CHEBI_CHMO_EXT:buffer_solution"},{"id":"T7164","span":{"begin":407,"end":411},"obj":"NCBITaxon:9925"},{"id":"T7165","span":{"begin":412,"end":415},"obj":"GO:0071735"},{"id":"T7166","span":{"begin":469,"end":475},"obj":"CHEBI_CHMO_EXT:buffer_solution"},{"id":"T7167","span":{"begin":479,"end":484},"obj":"CL_GO_EXT:cell"},{"id":"T7168","span":{"begin":516,"end":518},"obj":"PR_EXT:000006300"},{"id":"T7169","span":{"begin":532,"end":537},"obj":"UBERON:0001977"},{"id":"T7170","span":{"begin":582,"end":588},"obj":"CHEBI_CHMO_EXT:buffer_solution"},{"id":"T7171","span":{"begin":633,"end":640},"obj":"CHEBI_SO_EXT:molecular_label_or_mark_or_tag_process"},{"id":"T7172","span":{"begin":648,"end":656},"obj":"GO:0042571"},{"id":"T7173","span":{"begin":674,"end":679},"obj":"CL_GO_EXT:cell"},{"id":"T7174","span":{"begin":723,"end":729},"obj":"CHEBI_CHMO_EXT:buffer_solution"},{"id":"T7175","span":{"begin":752,"end":760},"obj":"CHEBI_EXT:poly_vinyl_alcohol"},{"id":"T7176","span":{"begin":788,"end":794},"obj":"UBERON:0002107"},{"id":"T7177","span":{"begin":800,"end":807},"obj":"PATO_UBERON_EXT:neonate_or_newborn"},{"id":"T7178","span":{"begin":808,"end":812},"obj":"NCBITaxon_UBERON_EXT:pup"},{"id":"T7179","span":{"begin":860,"end":876},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T7180","span":{"begin":933,"end":941},"obj":"CHEBI_EXT:nitrogen"},{"id":"T7181","span":{"begin":949,"end":959},"obj":"CHEBI:30362"},{"id":"T7182","span":{"begin":1024,"end":1029},"obj":"CL_GO_EXT:cell"}],"text":"Primary mouse embryonic fibroblasts were prepared from embryonic day ~16.5–18.5 fetuses as described [29]. Fibroblasts were passed onto glass, fixed in 2% paraformaldehyde in PBS for 10 minutes, permeabilized in 2% paraformaldehyde containing 0.1% Triton X100 for 10 minutes and washed three times in PBS containing 0.5% BSA and 0.15% glycine, pH 7.4 (Buffer A). Following a 30 min incubation with purified goat IgG (50 (μg/ml) at 25°C and three additional washes with Buffer A, cells were incubated for 60 min with E2-specific antiserum [30] at 1 μg/ml followed by three washes in Buffer A and 60 minute incubation in fluorescently labeled second antibody (1–2 μg/ml). The cells were then washed six times (5 min/wash) in Buffer A and then mounted in gelvatol (Monsanto, St Louis). When livers from newborn pups were examined, fixation was by immersion in 2% paraformaldehyde followed by cryoprotection and shock freezing in liquid nitrogen cooled isopentane and sectioning (5 microns). Otherwise processing was as for the cells above (without the fixation and permabilization steps)."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T7141","span":{"begin":8,"end":13},"obj":"NCBITaxon:10088"},{"id":"T7142","span":{"begin":14,"end":23},"obj":"UBERON:0000922"},{"id":"T7143","span":{"begin":24,"end":35},"obj":"CL:0000057"},{"id":"T7144","span":{"begin":55,"end":64},"obj":"UBERON:0000922"},{"id":"T7145","span":{"begin":107,"end":118},"obj":"CL:0000057"},{"id":"T7146","span":{"begin":248,"end":259},"obj":"CHEBI:9750"},{"id":"T7147","span":{"begin":407,"end":411},"obj":"NCBITaxon:9925"},{"id":"T7148","span":{"begin":412,"end":415},"obj":"GO:0071735"},{"id":"T7149","span":{"begin":516,"end":518},"obj":"PR:000006300"},{"id":"T7150","span":{"begin":532,"end":537},"obj":"UBERON:0001977"},{"id":"T7151","span":{"begin":648,"end":656},"obj":"GO:0042571"},{"id":"T7152","span":{"begin":788,"end":794},"obj":"UBERON:0002107"},{"id":"T7153","span":{"begin":949,"end":959},"obj":"CHEBI:30362"}],"text":"Primary mouse embryonic fibroblasts were prepared from embryonic day ~16.5–18.5 fetuses as described [29]. Fibroblasts were passed onto glass, fixed in 2% paraformaldehyde in PBS for 10 minutes, permeabilized in 2% paraformaldehyde containing 0.1% Triton X100 for 10 minutes and washed three times in PBS containing 0.5% BSA and 0.15% glycine, pH 7.4 (Buffer A). Following a 30 min incubation with purified goat IgG (50 (μg/ml) at 25°C and three additional washes with Buffer A, cells were incubated for 60 min with E2-specific antiserum [30] at 1 μg/ml followed by three washes in Buffer A and 60 minute incubation in fluorescently labeled second antibody (1–2 μg/ml). The cells were then washed six times (5 min/wash) in Buffer A and then mounted in gelvatol (Monsanto, St Louis). When livers from newborn pups were examined, fixation was by immersion in 2% paraformaldehyde followed by cryoprotection and shock freezing in liquid nitrogen cooled isopentane and sectioning (5 microns). Otherwise processing was as for the cells above (without the fixation and permabilization steps)."}